This invention pertains generally to the field of microwave spectroscopy and particularly to dielectric spectroscopy apparatus and to assays of proteins and other biological molecules.
Changes in the conformation of proteins in solution may occur for a variety of reasons, including ligand binding, enzyme activity, chemical or thermal denaturation and mutations or deletions. See, generally, T. E. Creighton, Proteins: Structures and Molecular Properties, 2nd Ed. New York, W. H. Freeman and Company, 1993. Most researchers use optical methods to observe such changes, such as ultraviolet-visible (UV/VIS), fluorescence, or circular dichroism spectroscopies. Such optical methods generally require high protein concentrations and large volume, but optical spectroscopy instrumentation is readily available and analysis with such instrumentation is not difficult. Other, less common methods include differential scanning calorimetry and electron paramagnetic resonance. Protein structure may be explicitly determined using nuclear magnetic resonance or x-ray diffraction, but these direct methods are time consuming, complex and require specialized facilities or equipment.
An alternative method for detection of protein conformational changes employs dielectric dispersion of water at frequencies in the microwave range. All proteins have low permittivity due to the arrangement of charged residues, including the N- and C-temini, xcex1-helices, and dipoles along the protein backbone. The static dielectric constant, xcex5xe2x80x2, of a typical protein has been estimated as 2 to 5 at room temperature. R. Pethig, Dielectric and Electronic Properties of Biological Materials, Chichester, John Wiley and Sons, 1979. Pure water possesses a much larger dielectric constant, which is approximately 80 at 25xc2x0 C. R. Pethig, ibid. All proteins are surrounded by one or more shells of xe2x80x9cboundxe2x80x9d water. Some proteins even have water molecules integrated into their structure. The presence of so much water hinders detection of the protein dielectric dispersion. However, this xe2x80x9cboundxe2x80x9d water may be distinguished from the water in bulk solution. In particular, the bound water undergoes dielectric dispersion at lower frequencies than water in bulk solutions. See, R. Pethig, xe2x80x9cProtein Water Interactions Determined by Dielectric Methods,xe2x80x9d Annu. Rev. Phys. Chem., Vol. 43, 1992, pp. 177-205. Bound water will be released or rearranged in response to the changes in protein conformation, leading to changes in the permittivity of the solution. Measurements of such dielectric dispersion have conventionally been performed using time domain spectroscopy (TDS), waveguides, or coaxial probes. See, Y. Feldman, et al., xe2x80x9cTime Domain Dielectric Spectroscopy: An Advanced Measuring System,xe2x80x9d Rev. Sci. Instrum., Vol. 67, 1996, pp. 3208-3216; G. R. Facer, et al., xe2x80x9cDielectric Spectroscopy for Bioanalysis: From 40 Hz to 26.5 GHz in a Microfabricated Wave Guide,xe2x80x9d Applied Physics Letters, Vol. 78, 2001, pp. 996-998; Y. Xu, et al., xe2x80x9cOn the Measurement of Microwave Permittivity of Biological Samples Using Needle-Type Coaxial Probes,xe2x80x9d IEEE Trans. Instrum. Meas., Vol. 42, 1993, pp. 822-827. TDS is by far the most common approach. TDS experiments involving protein conformational changes have been performed from 100 kHz to 10 GHz. Y. Feldman, et al., supra. TDS is not commonly used by biological scientists, possibly because of the complicated analysis that is required. Data must be converted from the time to the frequency domain, and then the response function must be transformed to complex permittivity.
In accordance with the present invention, dielectric spectroscopy is carried out by coupling microwave energy from the non-radiated field of an antenna to a sample solution to detect changes in permittivity of the sample within the antenna""s non-radiated field. The antenna and its associated drive circuitry and components exhibit a resonant frequency or frequencies in a frequency range of interest, typically in the range from 0.5 GHz to 50 GHz. The frequency response of the antenna as coupled to the sample is determined. Changes in the sample as a result of changes in environmental conditions of the sample that change the permittivity of the same within the antenna""s near zone will be manifested as changes in the magnitude or phase characteristics of the antenna""s resonant frequency or frequencies. The frequency response of the antenna may be determined at selected times corresponding to changed environmental conditions of the sample, allowing changes in the frequency response of the antenna to be correlated with the changed environmental conditions. By carrying out dielectric spectroscopy in this manner, data collection and analysis is significantly simplified. Data is collected in the frequency domain, eliminating the need to convert data from the time domain to the frequency domain. Explicit determination of complex permittivity is not necessary. Analysis of data obtained in accordance with the invention is no more complicated than analysis of conventional optical spectroscopy data.
The apparatus of the present invention includes an antenna mounted with a sample container in position to have its non-radiated microwave near field coupled to a sample held within the container. A preferred antenna is a resonant slot antenna, for example, having a circular or rectangular slot configuration. The dimensions of the slot can be selected to obtain the desired frequency range for the system, and such resonant slot antennas can be obtained commercially or manufactured economically from available materials. The window in the antenna provided by the slot allows passage of a light beam, facilitating the combination of dielectric spectroscopy in accordance with the invention with conventional optical spectroscopy. In this manner, simultaneous measurements of dielectric dispersion and other phenomena can be performed. Such antennas may be miniaturized and integrated into semiconductor chips, allowing antennas to be placed into environments that are not suitable for conventional optical measurements. An antenna, as utilized in the invention, may be any element which allows coupling of the non-radiated microwave field to a sample, and is not limited to conventional antenna structures.
Further objects, features and advantages of the invention will be apparent from the following detailed description when taken in conjunction with the accompanying drawings.